Preliminary investigations into the phylogenetic relationships in the genus Erica L.

Lester, Ntsikelelo Blessings (2006-12)

Thesis (MSc)--University of Stellenbosch, 2006.

Thesis

ENGLISH ABSTRACT: Erica is a genus of about 860 species world wide, with 700 of these found in South Africa’s southwestern and southern Cape, making it by far the most speciose genus in the Cape Floristic Region. This poses a particular challenge in the construction of a molecular phylogeny of the genus. The choice of suitably variable gene regions is a crucial decision on which the successful phylogenetic reconstruction of this important genus is critically dependent. The aim of this project was therefore to determine which DNA regions, both chloroplast and nuclear, would be sufficiently variable to give adequate informative characters that may be useful at the species level phylogenetic reconstruction. A subset of 30 species, representing the range of morphological diversity and pollinator preference within Erica, was selected for study. For each of these species the variability in eight chloroplast regions (trnL-F, matK, trnS-G, rps12- rpl20, psbAtrnH, trnC-D, rps4-trnT and trnT-L) and the nuclear ITS region was investigated. The psbA-trnH, trnC-D, rps4-trnT and trnT-L chloroplast regions were found to be problematic to amplify and to possess too few Parsimony Informative Characters to be of use in phylogenetic reconstruction. Four of the chloroplast regions, trnS-G, trnL-F, matK and rpS12-rpL20 and the nuclear ITS region could be amplified and sequenced with success. The ITS region was found to be reasonably variable, with the chloroplast genes showing less variability. The DNA extraction method employed showed itself to be of critical importance in the success of the study. Two DNA extraction protocols, both modified from the original Doyle and Doyle (1987) method, were tested. The one included double the amount of β-mercaptoethanol and Polyvinylpyrrolidone (PVP) and the other included an extended phenol: chloroform: isoamylalcohol step. These variables, together with the effectiveness of these methods on fresh vs. silica dried plant samples, were investigated to determine which of the two would yield high quantities and qualities of DNA and result in the best method for the extraction of DNA from Erica species.

AFRIKAANSE OPSOMMING: Erica is ‘n genus van omtrent 860 spesies wêreldwyd, met 700 van hierdie spesies aanwesig in die suidwes en suid Kaap van Suid Afrika, wat dit by verre die mees spesieryke genus in die Kaapse Floristiese Streek maak. Dit stel ’n besondere uitdaging in die konstruksie van ’n molekulêre filogenie van die genus. Die keuse van geskikte variërende geen-areas is ‘n belangrike besluit waarvan die suksesvolle filogenetiese rekonstruksie van hierdie belangrike genus krities afhanklik sal wees. Die doel van hierdie projek was dus om te bepaal watter DNS areas, buide chloroplas en kern, genoegsaam varieer om voldoende informatiewe kenmerke te lewer om bruikbaar te wees in ’n spesie-vlak molekulêre rekonstruksie. ’n Subgroep van 30 spesies, wat die reeks van morfologiese diversiteit en bestuiwer voorkeure in Erica verteenwoordig, is dus vir die studie geselekteer. Vir elk van hierdie spesies is die variasie in agt chloroplast areas (trnL-F, matK, trnS-G, rps12- rpl20, psbA-trnH, trnC-D, rps4-trnT en trnT-L) en die kern ITS area ondersoek. Dit was problematies om die psbA-trnH, trnC-D, rps4-trnT en trnT-L chloroplast areas te amplifiseer, en daar is gevind dat hulle te min Parsimonie Informatiewe Kenmerke besig om bruikbaar te wees in filogenetiese rekonstruksie. Vier van die chloroplas areas, trnS-G, trnL-F, matK en rpS12-rpL20 en die kern ITS kon suksesvol geamplifiseer word en die basisvolgordes kon suksesvol bepaal word. Daar is gevind dat die ITS area redelik variërend is, terwyl chloroplas areas minder variasie getoon het. Die DNS ekstraksie metode wat gebruik is het die kritiese belang van die ekstraksie metode in die sukses van die studie bewys. Twee DNS protokolle, beide gemodifiseer van die oorspronklike Doyle en Doyle (1987) metode, is getoets. Die een het dubbel die hoeveelheid β-mercaptoetanol en Polyvinylpyrrolidone (PVP) bevat, en die het ’n uitgebruide fenol: chloroform: isoamylalkohol stap ingesluit. Hierdie veranderlikes, saam met die effektiwiteit van hierdie metodes op vars teenoor silika-gedroogde plant monsters, is ondersoek om vas te stel watter een van die twee die hoogste kwaliteit en kwantiteit DNS sou lewer en dus sal lei tot die beste DNS ekstraksie metode vir Erica spesies.

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